d -Galactoside-Specific Lectins from the Body Wall of an Echiuroid (Urechis unicinctus) and Two Annelids (Neanthes japonica and Marphysa sanguinea)

Comp. Biochem. Physiol. Vol. 118B, No. 1, pp. 1–6, 1997 Copyright  1997 Elsevier Science Inc. All rights reserved.

ISSN 0305-0491/97/$17.00 PII S0305-0491(97)00014-X

d-Galactoside-Specific Lectins from the Body Wall of an Echiuroid (Urechis unicinctus) and Two Annelids (Neanthes japonica and Marphysa sanguinea) Yasuhiro Ozeki,† Eigoro Tazawa,† and Taei Matsui‡ †Department of System Element, Faculty of Science, Yokohama City University, Yokohama 236, Japan; and ‡Division of Biomedical Polymer Science, Institute for Comprehensive Medical Science, Fujita Health University, Toyoake 470-11, Japan ABSTRACT. Lectins recognizing d-galactosides were purified from the body wall of an echiuroid; Urechis unicinctus and two annelids; Neanthes japonica and Marphysa sanguinea, with single step lactosyl-agarose affinity column chromatography. SDS-PAGE under reduced and non-reduced conditions showed that U. unicinctus lectin had a major (36 kDa) and two minor (40 and 14 kDa) proteins, and that N. japonica lectin and M. sanguinea lectin had single 33 and 35 kDa proteins, respectively. Lectins were solubilized in the presence of lactose from tissues, and all polypeptides were shown to have sugar binding activity. The antisera raised against U. unicinctus lectin and N. japonica lectin crossreacted with each other but did not crossreact with bull frog (Rana catesbeiana) egg galectin-1 or a d-galactoside-specific lectin purified from sea urchin (Anthocidaris crassispina) eggs. These echiuroid and annelid lectins are immunologically similar, but distinct from members of the vertebrate galectin family. comp biochem physiol 118B;1:1–6, 1997.  1997 Elsevier Science Inc. KEY WORDS. Lectin, galectin, d-galactoside, affinity chromatography, immunocrossreactivity, echiuroid, annelid, preparative SDS-PAGE

INTRODUCTION d-Galactoside-specific lectins, which do not require divalent cations for their activity, are widely distributed among animal tissues. Many of the lectins discovered in tissues of vertebrates (3,8,18,21,23) are β-galactoside binding. They consist of several isolectins with similar primary structure and a typical molecular mass of 14 or 30 kDa. Animal lectins are currently classified as members of the ‘‘galectin super family’’ (4). Recently, the existence of a galectin family of proteins in not only vertebrates but also invertebrates such as nematoda, Caenorhabditis elegans (9,10) and sponge, Geodia cydonium (22) was reported. In addition, several lectins which recognize galactose and other specific oligosaccharides have been detected in invertebrates, for example, calcium-dependent (C-type) lectin in the coelomic fluid of

Address reprint requests to: Y. Ozeki, Department of System Element, Faculty of Science, Yokohama City University, 22-2 Seto, Kanazawaku, Yokohama, 236 Japan. Tel. 81(Japan)-45-787-2221; Fax 81(Japan)-45-7872370. Abbreviations–BFEL, bull frog egg galectin-1; BSA, bovine serum albumin; EDTA, ethylenediamine tetraacetic acid; HRP, horse radish peroxidase; UUL, Urechis unicinctus lectin; MSL, Marphysa sanguinea lectin; NJL, Neanthes japonica lectin; PAGE, polyacrylamide gel electrophoresis; SDS, sodium dodecyl sulfate; SUEL, sea urchin egg d-galactoside binding lectin; TBS, Tris-buffered saline. Received 22 August 1996; revised 3 January 1997; accepted 21 January 1997.

sea urchin (5) and sea cucumber (7,15), suggesting a defensive or cell adhesive function. A lectin consisting of a disulfied-linked homodimer of 11.5 kDa subunits which recognizes the anomeric structures of α- and β-galactosides was discovered in unfertilized eggs of the sea urchin, Anthocidaris crassispina (24). This lectin has a unique primary structure (19) and disappears after the pluteus stage (20). Its likely function is the regulation of morphogenesis during the early development of embryos (20). Another d-galactoside binding lectin, echinonectin, consisting of a disulfied-linked homodimer of glycosylated 116 kDa, was purified from the embryos of the sea urchin Lytechinus variegatus (1) as a cell adhesion protein, at the beginning of development (2). Matsui reported on 34/31 kDa d-galactoside binding lectins from coelomocytes of the echiuroid, Urechis unicinctus (14). Co-existence of a haptenic sugar was necessary for the extraction of the lectins from the coelomocytes, suggesting they exist with their endogenous ligand(s). Echiuroid is currently classified in the phylum of Annelida as Echiuroidae; phylogenically near to Polychaeta including annelids (6). Recently, a 30 kDa β-galactoside binding lectin was purified from the body of an annelid, Chaetopterus variopedatus, without adding haptenic sugars (16). A comparison of the lectins of these marine worms revealed molecular diversity between echiuroid and annelid. In this study, we attempted to purify the newly-discovered echiuroid and annelid lectins, which recognize d-galactoside sugar, from body wall and

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compared such traits as sugar binding, molecular mass, and immunocrossreactivity. MATERIALS AND METHODS Purification of Lectins from Echiuroid and Annelid Echiuroid worms, U. unicinctus collected at Choshi, Chiba Prefecture in Japan were opened by laparotomy, and the coelomic fluid and inner organs discarded. Annelid worms, Neanthes japonica and Marphysa sanguinea were purchased from a fishing shop. Their bodies were cut parallel to the axial line and the inner organs discarded. The body wall was homogenized with 10 vols (W/V) of 150 mM NaCl containing 20 mM Tris-HCl, pH 7.5 (TBS), 2 mM β-mercaptoethanol and 10 mM EDTA (buffer A). The precipitate obtained from centrifugation at 27,500 g for 1 hr at 4°C was resuspended with 3 vols of TBS containing 100 mM lactose (buffer B) and incubated at 4°C overnight. The supernatant obtained by further centrifugation was extensively dialyzed against 100 mM NaHCO 3 , pH 8.8 for 72 hr at 4°C. The dialyzate was then centrifuged at 27,500 g for 1 hr and the supernatant was applied to a lactosyl-agarose column (1.0 3 4.0 cm, Seikagaku-Kogyo, Tokyo, Japan). The column was washed with buffer A until the absorbance at 280 nm reached the baseline. The lectin was eluted with buffer B from the column and extensively dialyzed against buffer A. The protein concentration of the crude extract solution and the purified lectins was estimated by the method of Lowry et al. (12) using BSA as standard.

filter and centrifuged at 3,000 rpm for 5 min, then washed three times with TBS. The lower fraction was dialyzed against distilled water and concentrated into 50 µl. The upper fraction was solubilized with the same volumes of TBS as the lower fraction, then dialyzed and concentrated into 50 µl. The detection of the protein contained in each fraction was checked with SDS-PAGE. SDS-PAGE Purified echiuroid lectin was mixed with an equal volume of sample buffer (20 mM Tris-HCl, pH 6.8; 0.2% SDS, 2% β-mercaptoethanol; 20% glycerol). Aliquots of 35 µl were applied to the well of the mini-slab gel without heating. Preparative SDS-PAGE was performed with an electrode

Hemagglutination Assay A hemagglutination assay was performed using trypsinized and glutaraldehyde-fixed rabbit erythrocytes as described previously (14,24). Hemagglutination was measured in the presence of 0.025% Triton X-100 using microtiter V-plates and expressed as titer; defined as the reciprocal of the highest dilution giving positive hemagglutination. For the sugar inhibition assay, each sugar solution (200 mM) was serially diluted with TBS and mixed with lectin solution previously adjusted to titer 16. The minimum inhibitory sugar concentration was scored. The sugars were of specially pure grade, purchased from Wako Pure Chemicals (Osaka, Japan) and Sigma Chemicals (St Louis, MO, U.S.A.). Native Molecular Mass Estimation of the Purified Lectins Apparent molecular mass was estimated by gel filtration on a column packed with Sephacryl S-300 (1.0 3 90 cm, Pharmacia, Uppsala, Sweden) equilibrated with buffer B. The native molecular mass of each lectin was confirmed by membrane filtration with Centriflo CF 50A (Amicon, Denvers, MA, U.S.A.), which holds proteins above 50,000 Da. Lectin (ca. 30 µg of each) was loaded on the membrane

FIG. 1. Affinity purification of UUL, NJL and MSL. Lactose

extracts of body wall of an echiuroid (U. unicinctus) and two annelids (N. japonica and M. sanguinea) were applied to a lactose-conjugated agarose column equilibrated with buffer A after extensive dialysis. UUL, NJL, and MSL bound to the column were eluted with buffer B (arrow). Collection is indicated by a horizontal bar. (A) UUL; (B) NJL; and (C) MSL.

Purification of Lectins from Echiuroid and Annelids

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RESULTS Purification of Lectins from Echiuroid and Annelid Body Wall

FIG. 2. Hemagglutination activity of 40, 36, and 14 kDa

proteins in UUL. (A) After separation of each protein in UUL by the method of preparative SDS-PAGE, four columns of the gel were cut into strips and one of them was stained with Coomassie Blue to confirm the banding pattern. The remaining unstained strips, the area indicated by each closed triangle being equivalent to each polypeptide, were sliced and the protein eluted from the gel with TBS (3 vols. per weight of gel strip) overnight at 4°C. (B) Each eluted sample (40, 36, and 14 kDa proteins, respectively) was assayed for hemagglutination activity. a, b, and c designate the 40, 36, and 14 kDa proteins, respectively; L, assay with 50 mM lactose. Matrices and dots in wells indicate positive and negative hemagglutination, respectively.

TBS-extracts obtained from echiuroid and annelid body wall exhibited little hemagglutinating activity against rabbit erythrocytes (data not shown). However, the extensively dialyzed crude extracts, solubilized with TBS containing 100 mM lactose strongly agglutinated the erythrocytes. The hemagglutinating activities of the extracts were inhibited in the presence of lactose. Three lectins, one from echiuroid (U. unicinctus) and two from annelids (N. Japonica and M. sanguinea) were purified from the crude extracts using lactose-conjugated agarose affinity chromatography, as shown in Fig. 1A–C. Preparative SDS-PAGE pattern of the column eluate of U. unicinctus with buffer containing lactose produced one major (36 kDa) and two minor (40 and 14 kDa) bands under reducing conditions as shown in Fig. 2A. The banding pattern was identical in both preparative SDSPAGE and SDS-PAGE (cf. with Fig. 3, lane A). The three proteins, being equivalent to each band (40, 36, and 14 kDa), were eluted to buffer from unstained gel. All showed hemagglutination activity against rabbit erythrocytes; this activity was inhibited upon addition of lactose (Fig. 2B). Thus, all three proteins purified as U. unicinctus lectin (UUL) had lectin activity. The other two lectins; N. japonica lectin (NJL), and M. sanguinea lectin (MSL) showed single bands of 33 and 35 kDa, respectively, under reducing conditions, on SDS-PAGE (Fig. 3, lanes B and C). The

and gel buffer containing 0.1% SDS at a constant 50 volts at 4°C. SDS-PAGE (10% separation gel) was performed by the method of Laemmli (11). Preparation of Antisera and Western Blotting Antisera against echiuroid lectin and annelida, N. japonica lectin were raised in rabbits by repeated injection (in total, 1 mg each) of purified lectin emulsified with Freund’s adjuvant (Wako Pure Chemicals, Japan). Preimmune, antiechiuroid lectin, and anti-annelida lectin sera were stored in aliquots at 280°C until use. An echiuroid lectin and two annelid lectins were subjected to SDS-PAGE followed by electroblotting onto an Immobilon P membrane (Milipore, Bedford, MA) as described previously (13). The membrane was blocked with TBS containing 0.05% Tween-20 (buffer C), incubated with primary antisera diluted 1:500 with buffer C at room temperature for 2 hr, washed with buffer C, and then incubated with HRP-conjugated second goat anti-rabbit antibody (diluted 1 :1000) (MBL, Nagoya, Japan) for 1 hr. The HRP reaction was performed with 4chloro-1-naphthol and H 2O 2 as substrates.

FIG. 3. SDS-PAGE of lectins. Purified UUL (lanes A and D), NJL (lanes B and E), and MSL (lanes C and F) were subjected to SDS-PAGE under reducing (lanes A–C) and nonreducing (D–F) conditions, and stained with Coomassie Blue. The standard proteins used were: phosphorylase b (97 kDa), BSA (66 kDa), aldolase (42 kDa), carbonic anhydrase (30 kDa), trypsin inhibiter (20 kDa), and lysozyme (14 kDa).

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gesting that the major and minor components of these lectins exist as monomer and dimer subunits, respectively, in solution. The 14 kDa polypeptide in UUL is believed to exist as a non-covalently bound dimer because the 14 kDa component was eluted at a position that almost overlapped with the 36 and 40 kDa components of UUL by gel filtration (see Fig. 4A). This was confirmed by SDS-PAGE (data not shown). A summary of the purification of each lectin is shown in Table 1. In a typical purification experiment, 0.5 to 1.0 mg of lectin was purified from 100 g of fresh material. Characterization of the Lectins

FIG. 4. Estimation of native molecular mass of UUL, NJL,

and MSL. (A) The three purified lectins were subjected to gel filtration using a Sephacryl S-300 column (1.0 3 90 cm). Each lectin was eluted as a single peak corresponding to a molecular mass of about 35 kDa either in the absence or presence of 100 mM lactose. Alcohol dehydrogenase (150 kDa) (1), BSA (66 kDa) (2), carbonic anhydrase (29 kDa) (3), and cytochrome C (12.4 kDa) (4) were used as molecular mass standard markers. (B) Filtration of lectins with Centriflo CF50A membrane, which holds molecules greater than 50,000 Da. Upper (designated U) and lower (designated L) fractions were subjected to SDS-PAGE. (a) UUL; (b) NJL; and (c) MSL.

molecular mass of these lectins was slightly larger than that of the C. variopedatus lectin (30 kDa) reported by Mikheyskaya et al. (16). The SDS-PAGE profiles of each major band of the three lectins did not change between reducing and non-reducing conditions (Fig. 3A–C vs 3D–F), indicating that these lectins were not disulfied-linked multimers. All major lectins were eluted at a molecular mass of about 36,000 dalton by gel filtration on a Sephacryl S300 column (Fig. 4A). This was confirmed by membrane filtration. Almost all lectins loaded on the Centriflo CF 50A membrane passed though the membrane upon centrifugation (Fig. 4B, a–c, each of lane L), indicating that their native molecular masses were lower than 50 kDa, and sug-

The saccharide specificity of the three lectins is summarized in Table 2. The hemagglutinating activities of the lectins were inhibited by various d-galactosides. Thiodigalactoside was the most potent inhibitor followed by lactose. β-d-Galactosides (thiodigalactoside, lactose, and methyl β-d-galactopyranoside) were more effective than α-d-galactosides (melibiose and methyl α-d-galactopyranoside). However, the effect of the anomeric configuration was weaker than that of BFEL, a typical β-galactoside binding lectin (Table 2). The activity of each lectin was diminished by heat treatment at 70°C for 10 min but was not affected by the addition of either 50 mM EDTA or 10 mM β-mercaptoethanol (data not shown). Two rabbit antisera were raised against UUL and NJL. Both crossreacted with the 36 and 40 kDa components of UUL, NJA, and MSL (Fig. 5, lanes 4–6 vs 7–9). However, the antisera did not react with the 14 kDa component in UUL. Furthermore, neither antisera crossreacted with BFEL or SUEL (Fig. 5, lanes 14–17). DISCUSSION In this study, the three d-galactoside binding lectins (40, 36, and 14 kDa) newly elucidated to exist in the body wall of an echiuroid, U. unicinctus, were found to have the same biological properties as the 34/31 kDa d-galactoside binding lectins previously purified from the coelomocytes of the echiuroid (14); that is, they do not require divalent cation nor reducing reagent for the sugar binding activation and exist to be binding with endogenous ligands in tissue. How-

TABLE 1. Purification of the lectins from the body wall of echiuroid and annelids

Step Lactose extraction Affinity purification

Sample

Titer

Protein (mg/ml)

Volume (ml)

Total activity*

Specific activity**

Recovery (%)

U. unichinctus N. japonica M. sanguinea U. unichinctus N. japonica M. sanguinea

256 256 68 2048 2048 512

2.88 3.26 5.62 0.33 0.38 0.25

100 100 100 3.0 2.8 2.0

25,600 25,600 6,800 6,144 5,734 1,024

79 44 12 6,206 5,389 2,048

100 100 100 24 22 15

*Total activity; Titer 3 volume. **Specific activity; titer/mg of protein.

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TABLE 2. Effects of saccharides on lectin induced hemagglutination

Lectins UUL Saccharide Thiodigalactoside Lactose Methyl β -d-galactopyranoside Methyl α-d-galactopyranoside Melibiose Sucrose d-Galactose N-Acetyl-d-galactosamine N-Acetyl-d-glucosamine d-Mannose d-Glucose l-Fucose

NJL

MSL

BFEL*

Minimum inhibitory concentration (mM) 0.4 0.4 0.8 1.6 3.1 50.0 3.1 25.0 .50.0 .50.0 .50.0 .50.0

0.4 0.8 0.8 1.6 3.1 .50.0 1.6 25.0 .50.0 .50.0 .50.0 .50.0

0.4 0.4 0.8 1.6 1.6 .50.0 1.6 50.0 .50.0 .50.0 .50.0 .50.0

0.6 1.0 N.D.** N.D. 50.0 N.D. 50.0 50.0 .75.0 N.D. .75.0 N.D.

Lectin titer was previously adjusted to 16. Trypsinized and glutaraldehyde-fixed rabbit erythrocytes were used for the assay. *Taken from Ozeki et al. (19). **Not determined.

FIG. 5. Immunoblotting of echiuroid and annelid lectins

against anti-lectin sera. UUL (lanes 1, 4, 7, and 10), NJL (lanes 2, 5, 8, and 11), MSL (lanes 3, 6, and 9), BFEL (18) (lanes 12, 14, and 16), and SUEL (19) (lanes 13, 15, and 17) were transferred to a PVDF membrane after SDS-PAGE. Each column was stained with Coomassie blue (CBB, lanes 1–3, and 12–13), or immunoblotted with anti-UUL sera (lanes 4–6 and 14–15), anti-NJL sera (lanes 7–9 and 16– 17), and preimmune serum (pre., lanes 10 and 11).

ever, both the minor and major components (40 and 36 kDa) of UUL appeared to have a slightly larger molecular mass than the coelomocyte lectins. Also the relationship between the two component of these lectins was reversed, that is the lower molecular mass protein (36 kDa) was the major component in UUL, while the molecule with the higher molecular mass (34 kDa) was the major component in coelomocyte lectins. Thus, the 40 and 36 kDa components in UUL appear to be a different molecule species to the 34/31 kDa coelomocyte lectins, suggesting that several isolectins exist in U. unicinctus. The results from membrane and gel filtration analysis suggested that the 40 and 36 kDa components in UUL exist as monomers in solution and have more than two carbohydrate recognition domains in one polypeptide, like galectin-4 (9). Monomeric 33 and 35 kDa lectins similar to the major 36 kDa component of UUL were purified from annelids (as NJL and MSL, respectively), suggesting that monomeric 30–40 kDa d-galactoside binding lectins are widespread among this phylum. The d-galactoside-specific lectins (33–40 kDa and 14 kDa) purified from these Annelida resemble galectin families in terms of molecular mass and in that they solubilize with a haptenic sugar. However, they differ from the latter in sugar recognition specificity against not only β-galactosides but also α-galactosides such as melibiose. Furthermore, the antisera raised against UUL did not crossreact with lower vertebrate galectin-1. Recently a unique Ca 21-independent lectin was purified from the globiferous pedicallariae of the sea urchin Toxopneustes pileolus (17). The lectin, named SUL-1 consisted of a 32 kDa monomeric polypeptide and was reported to have high affinity against d-galactose. The biochemical properties of SUL-1 are similar to those of the major 36 kDa component of UUL, NJL, and MSL, suggesting the existence of an unique lectin family in ma-

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rine invertebrates. Structural analysis should elucidate whether such proteins represent the discovery of a new lectin family in invertebrates. This work was supported by Grants-in Aid from Yokohama City University.

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